Strain TA1 Research Articles

Whole genome analysis of Bacillus amyloliquefaciens TA-1, a promising biocontrol agent against Cercospora arachidicola pathogen of early leaf spot in Arachis hypogaea L

BackgroundEarly leaf spot disease, caused by Cercospora arachidicola, is a devastating peanut disease that has severely impacted peanut production and quality. Chemical fungicides pollute the environment; however, Bacillus bacteria can be used as an environmentally friendly alternative to chemical fungicides. To understand the novel bacterial strain and unravel its molecular mechanism, De novo whole-genome sequencing emerges as a rapid and efficient omics approach.ResultsIn the current study, we identified an antagonistic strain, Bacillus amyloliquefaciens TA-1. In-vitro assay showed that the TA-1 strain was a strong antagonist against C. arachidicola, with an inhibition zone of 88.9 mm. In a greenhouse assay, results showed that the TA-1 strain had a significant biocontrol effect of 95% on peanut early leaf spot disease. De novo whole-genome sequencing analysis, shows that strain TA-1 has a single circular chromosome with 4172 protein-coding genes and a 45.91% guanine and cytosine (GC) content. Gene function was annotated using non-redundant proteins from the National Center for Biotechnology Information (NCBI), Swiss-Prot, the Kyoto Encyclopedia of Genes and Genomes (KEGG), clusters of orthologous groups of proteins, gene ontology, pathogen-host interactions, and carbohydrate-active enZYmes. antiSMASH analysis predicted that strain TA-1 can produce the secondary metabolites siderophore, tailcyclized peptide, myxochelin, bacillibactin, paenibactin, myxochelin, griseobactin, benarthin, tailcyclized, and samylocyclicin.ConclusionThe strain TA-1 had a significant biological control effect against peanut early leaf spot disease in-vitro and in greenhouse assays. Whole genome analysis revealed that, TA-1 strain belongs to B. amyloliquefaciens and could produce the antifungal secondary metabolites.

The effect of probiotics on digestive enzyme activity during larvae and juvenile stage of Yellow Fin Tuna (Thunnus albacares)

Yellow fin tuna fry production technology is not successful yet. This was assumed related to the biological and physiological characteristics of the fish. The physiological approach to regulating digestion through probiotics is needs to be studied. The aim of this study was to examine the effect of probiotics bacterial strains of the enzymatic activity in digestion system for the larvae to juvenile stages of yellow fin tuna, Thunnus albacares. The experiment was initiated by culturing 3 probiotic isolates (Bacillus subtilis strain TA-1, Bacillus amyloliquefaciens strain TN-2, Bacillus subtilis strain TO-4) and apply to the larval rearing of yellow fin tuna. In the experiment the treatments applied were supplemented probiotics and without probiotic, each treatment was held with 2 replications. Fish samples were collected every day and analyze the gene expression profile associated with digestion enzyme synthesis by total RNA isolation and cDNA synthesis followed analysis of its by using the RT –qPCR method. The results showed that the growth response of T. albacares tuna larvae reared with probiotics tended to be faster (P<0.05) than the control (without probiotics). The supplementation of probiotic could improved the expressed digestibility of the target genes associated with enzymatic synthesized. Amylase enzymatic synthesized increase 200-1200 times in larvae 10 –17 Day After Hatch (DAH), while in the lipase enzymatic activity started with 13 DAH ( 25 time) and 16 – 21 DAH by 150-300 times. In trypsin the highest enzymatic activity was at 16 DAH, which was 200 times. While in control, the expression of enzymatic synthesis of amylase, lipase, and trypsin was relatively low.

Cross-host compatibility of commercial rhizobial strains for new and existing pasture legume cultivars in south-eastern Australia

Perennial legumes have potential to increase pasture productivity in the high rainfall zone (600–850 mm) of south-eastern Australia through their ability to use summer rainfall and fix nitrogen (N2). Various perennial legumes are being evaluated for this environment; however, little information exists on legume–rhizobia cross-host compatibility and its consequences for biological N2 fixation. This is especially important when legumes are sown into fields with a background of competitive rhizobia such as WSM1325 or sown as a pasture mix with different host–symbiont pairs. We studied the effectiveness and cross-host compatibility of five commercial rhizobial strains for a range of pasture legumes (nine species, 18 cultivars) under controlled environment conditions, and further evaluated nodule occupancy and competitiveness of a newly established pasture (13 species, 20 cultivars) in the field, by determining nodulation and production (biomass and N2 fixation). Three of the commercial inoculant strains formed root nodules with multiple legume species; commonly however, less N2 was fixed in cases where the inoculant was not the recommended strain for the legume species. Within a legume species, cultivars could differ in their ability to form effective root nodules with multiple rhizobial strains. White clover cvv. Trophy, Haifa and Storm, strawberry clover cv. Palestine, and Talish clover cv. Permatas formed effective nodules with both TA1 and WSM1325 rhizobial strains. White clover cultivars that could not form an effective symbiosis with the common background strain WSM1325 fixed less N2. The white clover × Caucasian clover hybrid formed effective symbiosis with strain TA1 but not with other commercial strains. Some species such as birdsfoot trefoil, Talish clover, sulfur clover and tetraploid Caucasian clover formed ineffective symbiosis in the field. Until resolved, this will likely inhibit their further development as pasture plants for similar permanent pasture environments.

Rhizobium leguminosarum bv. trifolii NodD2 Enhances Competitive Nodule Colonization in the Clover-Rhizobium Symbiosis.

Establishment of the symbiotic relationship that develops between rhizobia and their legume hosts is contingent upon an interkingdom signal exchange. In response to host legume flavonoids, NodD proteins from compatible rhizobia activate expression of nodulation genes that produce lipochitin oligosaccharide signaling molecules known as Nod factors. Root nodule formation commences upon legume recognition of compatible Nod factor. Rhizobium leguminosarum was previously considered to contain one copy of nodD; here, we show that some strains of the Trifolium (clover) microsymbiont R. leguminosarum bv. trifolii contain a second copy designated nodD2. nodD2 genes were present in 8 out of 13 strains of R. leguminosarum bv. trifolii, but were absent from the genomes of 16 R. leguminosarum bv. viciae strains. Analysis of single and double nodD1 and nodD2 mutants in R. leguminosarum bv. trifolii strain TA1 revealed that NodD2 was functional and enhanced nodule colonization competitiveness. However, NodD1 showed significantly greater capacity to induce nod gene expression and infection thread formation. Clover species are either annual or perennial and this phenological distinction is rarely crossed by individual R. leguminosarum bv. trifolii microsymbionts for effective symbiosis. Of 13 strains with genome sequences available, 7 of the 8 effective microsymbionts of perennial hosts contained nodD2, whereas the 3 microsymbionts of annual hosts did not. We hypothesize that NodD2 inducer recognition differs from NodD1, and NodD2 functions to enhance competition and effective symbiosis, which may discriminate in favor of perennial hosts.IMPORTANCE Establishment of the rhizobium-legume symbiosis requires a highly specific and complex signal exchange between both participants. Rhizobia perceive legume flavonoid compounds through LysR-type NodD regulators. Often, rhizobia encode multiple copies of nodD, which is one determinant of host specificity. In some species of rhizobia, the presence of multiple copies of NodD extends their symbiotic host-range. Here, we identified and characterized a second copy of nodD present in some strains of the clover microsymbiont Rhizobium leguminosarum bv. trifolii. The second nodD gene contributed to the competitive ability of the strain on white clover, an important forage legume. A screen for strains containing nodD2 could be utilized as one criterion to select strains with enhanced competitive ability for use as inoculants for pasture production.

Increasing biological nitrogen fixation by white clover-rhizobia symbiosis

Biological nitrogen fixation (BNF) is the process of converting atmospheric nitrogen to ammonia through legume–rhizobia symbiosis. The nitrogen fixed by rhizobia in root nodules is available for plant use. This process can be harnessed to improve N fertility on farm. Field surveys across New Zealand (NZ), within a farm and within paddocks, have revealed large spatial variability of rhizobial population size and symbiotic effectiveness with white clover. These results indicate that naturalised rhizobia may not be supporting optimal BNF. Over 500 strains of clover-nodulating rhizobia were isolated from NZ pasture soils, with more than 90 demonstrating greater N-fixation capacity with white clover than the commercial inoculant strain TA1. Seven NZ isolates were tested for nodule occupancy and all seven had significantly higher occupancy rates than TA1 in an in vitro assay, indicating increased competitiveness of those strains. In addition, novel seed-coating technology improved the survival of TA1 and isolate S10N9 from 1 month to more than 4 months compared with a standard coating formulation. There is potential to increase the symbiotic capacity of white clover in pastures through use of more effective and competitive rhizobial strains, along with their improved survival on seed provided by a new coating technology.

Symbiosis between Rhizobium leguminosarum bv. trifolii strain TA1 and a white clover cultivar benefits clover tolerance to cadmium toxicity

ABSTRACT New Zealand pastoral productivity relies heavily on biological nitrogen (N) fixation from rhizobia bacteria. Cadmium (Cd), present in pasture soils as an impurity in phosphate fertilisers, may have toxic effects on both plant growth and rhizobia activity. Effects of Cd on growth of seedlings of a white clover cultivar ‘Grasslands Tribute’, in the absence and presence of the N2-fixing Rhizobium leguminosarum bv. trifolii strain TA1, were assessed in vermiculite microcosm assays. The lowest observable effect concentrations resulting in significantly lower white clover shoot weights than those grown without Cd were 0.07 mg Cd kg−1 (measured as Ca(NO3)2-extractable Cd, Cdextractable) in the absence of Rhizobium, and 2.33 mg Cdextractable kg−1 for Rhizobium-inoculated seedlings. Shoot N content was unaffected in white clover exposed to <5 mg Cdextractable kg−1. The results indicate symbiosis with Rhizobium strain TA1 conferred tolerance to white clover ‘Grasslands Tribute’ against Cd toxicity, and that this symbiosis is unlikely to be adversely affected by Cd concentrations reported (<0.003 mg Cdextractable kg−1) in New Zealand pasture soils.

English

Studies on actinobacteria of marine habitats are gaining international importance due to proven abilities of novel compound production. Information on actinobacteria of the marine environments is less and hence the present studies were optimum at higher temperature, which indicates that they are thermophilic in nature. It can be conducted that the two strains (TA-1 and TA-2) belong to two different genera viz. Rhodococcus (TA-1) and Streptosporangium (TA-2). Actinobacterial strains were isolated from the sediment samples collected from the mud volcano, of the Baratang Island andaman and Nicobar group of islands. Out of a total of six isolates, two different actinobacterial strains (TA-1 and TA-2) were selected based on the morphological distinct colour of spore mass, riverside colour, arial and mycelia format production of diffusible pigment sporophore and colony morphology. Morphological studies indicated that the strains belonged to two different genera viz. Rhodococcus (TA-1) and Streptosporangium (TA-2). The strain had no aerial mycelium and analysis of the strain TA-1 showed fragmented rods, filaments and hyphae, and the strain TA-2 showed that the formed globosa sporangia on the surface of the colony liberated non-motile spores. Key words: Thermophilic actinobacteria, isolation and identification, Baratang Island.

Genome sequence of the clover-nodulating Rhizobium leguminosarum bv. trifolii strain TA1

Rhizobium leguminosarum bv. trifolii strain TA1 is an aerobic, motile, Gram-negative, non-spore-forming rod that is an effective nitrogen fixing microsymbiont on the perennial clovers originating from Europe and the Mediterranean basin. TA1 however is ineffective with many annual and perennial clovers originating from Africa and America. Here we describe the features of R. leguminosarum bv. trifolii strain TA1, together with genome sequence information and annotation. The 8,618,824 bp high-quality-draft genome is arranged in a 6 scaffold of 32 contigs, contains 8,493 protein-coding genes and 83 RNA-only encoding genes, and is one of 20 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Community Sequencing Program.

Biological activity of (Lipo)polysaccharides of the exopolysaccharide-deficient mutant Rt120 derived from Rhizobium leguminosarum bv. trifolii strain TA1

Lipopolysaccharides (LPS) from Rhizobium leguminosarum biovar trifolii TA1 (RtTA1) and its mutant Rt120 in the pssBpssA intergenic region as well as degraded polysaccharides (DPS) derived from the LPS were elucidated in terms of their chemical composition and biological activities. The polysaccharide portions were examined by methylation analysis, MALDI-TOF mass spectrometry, and (1)H NMR spectroscopy. A high molecular mass carbohydrate fraction obtained from Rt120 DPS by Sephadex G-50 gel chromatography was composed mainly of L-rhamnose, 6-deoxy-L-talose, D-galactose, and D-galacturonic acid, whereas that from RtTA1 DPS contained L-fucose, 2-acetamido-2,6-dideoxy-D-glucose, D-galacturonic acid, 3-deoxy-3-methylaminofucose, D-glucose, D-glucuronic acid, and heptose. Relative intensities of the major (1)H NMR signals for O-acetyl and N-acetyl groups were 1 : 0.8 and 1 : 1.24 in DPS of Rt120 and RtTA1, respectively. The intact mutant LPS exhibited a twice higher lethal toxicity than the wild type LPS. A higher in vivo production of TNFα and IL-6 after induction of mice with Rt120 LPS correlated with the toxicity, although the mutant LPS induced the secretion of IL-1β and IFNγ more weakly than RtTA1 LPS. A polysaccharide obtained by gel chromatography on Bio-Gel P-4 of the high molecular mass material from Rt120 had a toxic effect on tumor HeLa cells but was inactive against the normal human skin fibroblast cell line. The polysaccharide from RtTA1 was inactive against either cell line. The potent inhibitory effect of the mutant DPS on tumor HeLa cells seems to be related with the differences in sugar composition.

Purification and characterization of vanillin dehydrogenases from alkaliphileMicrococcussp. TA1 and neutrophileBurkholderia cepaciaTM1

Vanillin dehydrogenases (VDHs) were purified and characterized from two bacterial strains that have different pH dependencies for growth. The alkaliphile Micrococcus sp. TA1, isolated from an alkaline spa, can grow on several aromatic compounds such as ferulic acid, vanillin, vanillic acid, and protocatechuic acid under alkaline conditions. The neutrophile Burkholderia cepacia TM1, which was isolated previously, also grew on the above-mentioned compounds because they functioned as the sole carbon source under neutral conditions. Purified VDHs showed activities toward some aromatic aldehydes. These enzymes have the same subunit molecular mass of about 57 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but differed in some of their observed properties. Native molecular masses also differed between the purified enzymes. These were 250 kDa for the enzyme from alkaliphilic strain TA1 and 110 kDa for that from neutrophilic strain TM1, as determined by gel filtration. The enzyme from strain TA1 required NADP(+) as a coenzyme for its activity, but that from strain TM1 required NAD(+). These results are important because this is the first report of an alkaliphilic bacterium consuming lignin monomers.

Lipoprotein PssN of Rhizobium leguminosarum bv. trifolii: Subcellular Localization and Possible Involvement in Exopolysaccharide Export

Surface expression of exopolysaccharides (EPS) in gram-negative bacteria depends on the activity of proteins found in the cytoplasmic membrane, the periplasmic space, and the outer membrane. pssTNOP genes identified in Rhizobium leguminosarum bv. trifolii strain TA1 encode proteins that might be components of the EPS polymerization and secretion system. In this study, we have characterized PssN protein. Employing pssN-phoA and pssN-lacZ gene fusions and in vivo acylation with [3H]palmitate, we demonstrated that PssN is a 43-kDa lipoprotein directed to the periplasm by an N-terminal signal sequence. Membrane detergent fractionation followed by sucrose gradient centrifugation showed that PssN is an outer membrane-associated protein. Indirect immunofluorescence with anti-PssN and fluorescein isothiocyanate-conjugated antibodies and protease digestion of spheroplasts and intact cells of TA1 provided evidence that PssN is oriented towards the periplasmic space. Chemical cross-linking of TA1 and E. coli cells overproducing PssN-His6 protein showed that PssN might exist as a homo-oligomer of at least two monomers. Investigation of the secondary structure of purified PssN-His6 protein by Fourier transform infrared spectroscopy revealed the predominant presence of beta-structure; however, alpha-helices were also detected. Influence of an increased amount of PssN protein on the TA1 phenotype was assessed and correlated with a moderate enhancement of EPS production.

A novel butenoate derivative from Aspergillus niger

A novel butenoate derivative, methyl (Z)-4-{[(Z)-1-(hydroxymethyl)-2-phenyl-1-ethenyl] amino}-4-oxo-2-butenoate (1), was isolated from the fermentation broth of an Aspergillus niger strain Ta1. The structure (1) was elucidated by spectral analysis. Bioassays showed that compound 1 had a weak antioxidant activity.

Topological and transcriptional analysis of pssL gene product: a putative Wzx-like exopolysaccharide translocase in Rhizobium leguminosarum bv. trifolii TA1

An identified pssL gene is yet another one, besides the pssT, pssN and pssP genes, encoding for a protein engaged in polysaccharide polymerization and export in Rhizobium leguminosarum bv. trifolii strain TA1 (RtTA1). Amino acid sequence similarity and hypothetical protein secondary structure placed the PssL protein within Wzx (RfbX) translocases with putative flippase function that belong to the polysaccharide specific transport (PST) family. The predicted secondary structure of the PssL membrane protein was examined with a series of PssL-PhoA and PssL-LacZ translational fusions. The results support the hypothesis of PssL being a member of PST protein family comprising transporters with 12 membrane spanning segments and amino and carboxyl termini located in the cytoplasm. Results of semi-quantitative RT-PCR showed that the initial abundance of mRNA encoding PssL protein was relatively lower when compared to the quantity of the previously identified PssT membrane protein. PssL might be a good candidate for Wzx-like protein that together with PssT (Wzy protein) could be responsible for Wzx/Wzy-like-dependent EPS polymerization and translocation in RtTA1.

Complexity of phenotypes and symbiotic behaviour of Rhizobium leguminosarum biovar trifolii exopolysaccharide mutants.

Rhizobium leguminosarum biovar trifolii strain TA1 polysaccharide synthesis (pss) mutants in the pssD, pssP, pssT and pssO genes and altered in exopolysaccharide (EPS) synthesis were investigated. EPS-deficient mutants were also changed in lipopolysaccharide structure. All mutants exhibited varied sensitivities to detergents, ethanol and antibiotics, thus indicating changes in bacterial membrane integrity. Using pss mutants marked with the gusA gene, EPS-deficient mutants were found to have abnormalities in nodule development and to provoke severe plant defence reactions. The pss mutants that produced altered quantities of EPS with a changed degree of polymerisation generally occupied the younger developmental zones of the nodules and elicited moderate plant defence reactions.

Competition between inoculant and naturalised Rhizobium leguminosarum bv. trifolii for nodulation of annual clovers in alkaline soils

Inoculant rhizobia typically need to compete with naturalised soil populations of rhizobia to form legume nodules. We have used the polymerase chain reaction to test the ability of seed-inoculated rhizobia to compete with naturalised populations of rhizobia and form nodules on clover (Trifolium alexandrinum, T.�purpureum, and T. resupinatum) in alkaline soil. Clover rhizobia, Rhizobium leguminosarum bv. trifolii, were identified at the strain level using either a nif-specific RP01 primer or ERIC primers. Analysis of rhizobia isolated from nodules indicated that strain TA1 competed poorly for nodule occupancy at 2 field sites (Roseworthy and Mallala, South Australia), with the exception that it nodulated T. alexandrinum at a level of 39% at the Roseworthy site in the first year of the trial. Strains CC2483g and WSM409 successfully colonised nodules when inoculated onto a particular clover species (T. resupinatum and T. purpureum, respectively) in the first year of inoculation and persisted in the soil to form nodules in the following year. Nodules frequently contained naturalised strains of rhizobia, distinct from introduced commercial strains. Dominant isolates were specific to a field site and nodulated all 3 clover species in both years of the field trial, with each isolate occupying up to 19% of the total nodules at a field site. It was hypothesised that field isolates had a better alkaline soil tolerance conferring a greater ability to nodulate clovers under these edaphic conditions. The results indicate that soil populations of rhizobia may provide a significant constraint to the introduction of current Australian commercial clover rhizobia into alkaline soils, and a more profitable strategy may be to seek rhizobial inoculants that are adapted to these soils.

The Rhizobium leguminosarum bv. trifolii pssB gene product is an inositol monophosphatase that influences exopolysaccharide synthesis.

The pssB gene of Rhizobium leguminosarum bv. trifolii encodes a protein of 284 amino acids with sequence similarity to eukaryotic inositol monophosphatases. The gene was cloned and overexpressed in Escherichia coli. The purified gene product of pssB showed inositol monophosphatase activity with a Km of 0.23 mM, and a Vmax of 3.27 mumol Pi min-1 (mg protein)-1. Its substrate specificity, Mg+2 requirement, Li+ inhibition, and subunit association (dimerization) were studied and compared to those of other inositol monophosphatases. Western immunoblotting with anti-PssB antibodies showed the presence of PssB in R. leguminosarum bv. trifolii strain TA1 and lack of this protein in the pssB mutant strain Rt12A. The presence of PssB protein in R. leguminosarum bv. trifolii TA1 was correlated with phosphatase activity with myo-inositol 1-phosphate as a substrate. Evidence for a regulatory function of PssB protein in exopolysaccharide (EPS) synthesis is presented. The mutation in pssB caused EPS overproduction, and introduction of pssB into the wild-type TA1 strain reduced EPS synthesis. The changes in the level of EPS production were correlated with a non-nitrogen-fixing phenotype of rhizobia.

Mutation in the pssB-pssA intergenic region of Rhizobium leguminosarum bv. trifolii affects the surface polysaccharides synthesis and nitrogen fixation ability

Summary A Rhizobium leguminosarum bv. trifolii Tn 5 transposon mutant deficient in exopolysaccharide biosynthesis was found to form non-nitrogen fixing nodules on clover. Root nodules induced by the mutant contained aberrant infection threads and few bacteroids. Sequence analysis of the transposon insertion site localized the mutation in the pssB-pssA intergenic region affecting the exopolysaccharide biosynthesis. The mutant also showed decreased sensitivity to SDS and deoxycholate and displayed a changed lipopolysaccharide (LPS) banding pattern compared to the wild-type strain TA1. The alteration in the O-polysaccharide part of LPS was confirmed by Western immunoblotting with polyclonal antibodies. LPS preparations of strain TA1 and the mutant strain only reacted with their homologous sera. The common epitopes in LPS from bacteroids and free-living rhizobia were revealed by immunogold assay. The results of this study indicate that the pssB-pssA region of R. leguminosarum bv. trifolii is important for the polysaccharide synthesis.

Molecular characterization and symbiotic importance of prsD gene of Rhizobium leguminosarum bv. trifolii TA1.

The prsD, prsE and orf3 genes of Rhizobium leguminosarum bv. trifolii strain TA1 encode the proteins which are significantly related to the family of bacterial ABC transporters type I secretion systems. The prsD:Km(r) mutant of strain TA1 induced non-nitrogen-fixing nodules on Trifolium pratense. Microscopic analysis of the nodules induced by prsD mutant did not reveal major abberations in the bacteroid appearance. The exopolysaccharide of prsD mutant was produced in increased amount and its level of polymerization was changed. SDS/PAGE of the proteins from the culture supernatants showed a lack of the 47-kDa protein in the culture of prsD mutant. Thus, PrsD may play a role in the export of this protein.

Molecular characterization of pssCDE genes of Rhizobium leguminosarum bv. trifolii strain TA1: pssD mutant is affected in exopolysaccharide synthesis and endocytosis of bacteria.

We have identified the three genes pssCDE in Rhizobium leguminosarum bv. trifolii TA1. Even though they were almost identical to earlier identified pssCDE genes of R. leguminosarum, they differed in gene lengths and gene overlaps. The predicted gene products of pssCDE genes shared significant homology to prokaryotic glycosyl transferases involved in exopolysaccharide synthesis. The Tn5 insertion in pssD created the nonmucoid mutant that induced non-nitrogen-fixing nodules. The microscopic analysis of the nodules, induced on Trifolium pratense by the pssD133 mutant, showed abnormally enlarged infection threads densely packed with bacteria, which were released from the infection threads in an unusual way. The symbiosomes were observed very rarely and the nodule remained almost empty. Symbiotic phenotype of the pssD133 suggested a correlation between this mutation and defective endocytosis of bacteria into nodule cells.

Response of intracellular carbohydrates to a NaCl shock in Rhizobium leguminosarum biovar trifolii TA-1 and Rhizobium meliloti SU-47

The dynamic response of cellular carbohydrates to a NaCl shock in Rhizobium leguminosarum biovar trifolii TA-1 (0·25 m-NaCl) and Rhizobium meliloti SU-47 (0·4 m-NaCl) grown in NaCl-free medium was investigated in non-growing cell cultures and in cell suspensions, using in vivo NMR. After transferring cells grown in a NaCl-free medium to a glutamic-acid-free medium containing mannitol and NaCl, both strains immediately responded to the increased osmotic pressure by augmenting the cellular trehalose content of the cell. Without mannitol in the medium trehalose synthesis was slower, but clearly detectable. Its synthesis paralleled the breakdown of the reserve materials glycogen and poly-β-hydroxybutyric acid (PHB). NMR experiments with 25-fold-concentrated cell suspensions using 13C1-mannitol as substrate revealed that 15–20% of the trehalose synthesized was derived from mannitol, but 80–85% was from other sources. Trehalose was mainly formed from the internal pool of glycogen and/or PHB, whether mannitol was present or not, and reached 135 and 280 μg (mg cell protein)−1 in the strains TA-1 and SU-47, respectively. At low osmolarity, intracellular trehalose was metabolized by strains TA-1 and SU-47. Intracellularly accumulated phosphoglycerol-substituted and neutral cyclic (1,2)-β-glucans of SU-47 cells grown in the absence of NaCl were neither degraded nor excreted after exposure to NaCl. Strain TA-1, which only makes neutral cyclic (1,2)-β-glucans, continued to synthesize and excrete cyclic (1,2)-β-glucans after exposure to NaCl. By using in vivo 31P-NMR, a sharp peak at 1·34 p.p.m. was present in cell suspensions of strain SU-47. This peak, representing glycerol-1-phosphate-substituted cyclic glucans, was absent in strain TA-1.